Peptide hormones secreted by enteroendocrine cells diffusely distributed as isolated cells along the mucosa of the GI tract orchestrate to regulate food initiation, digestion, gut motility and satiation. Along with other hormones released from pancreas, adipose tissue and the central nervous system, these gut hormones help to maintain body weight, glucose and energy homeostasis. Among the many GI peptide hormones, ghrelin, a 28-amino acid peptide with a Ser3 n-octanoylation, is secreted from mucosal enteroendocrine cells throughout the GI tract with a cell density that is highest in the gastric mucosa and declines caudally along the intestine. Consistent with the distribution of ghrelin cell density, 65 to 80% of circulating ghrelin is released from the stomach. Two types of ghrelin cells exist in the GI tract, a closed-type in the oxyntic mucosa of the stomach with no luminal exposure and an open-type with apical luminal contact in the remainder of the gut. These two types of ghrelin cells suggest that ghrelin secretion may be differentially regulated and that ghrelin may play different physiological roles in various regions of the GI tract. The physiological functions of ghrelin have been extensively studied. In both humans and other mammals, ghrelin levels rise before a meal fall rapidly after ingestion. On the basis of this phasic release, ghrelin has been speculated to play a short term role in food initiation. In long-term regulation of body weight, circulating ghrelin levels correlate inversely with body weight change. Chronic administration of ghrelin increases body weight and weight loss resulting from multiple causes is associated with elevated ghrelin levels. Similar to leptin and insulin, ghrelin has been postulated to be an adiposity signal. Currently, regulation of ghrelin secretion is being intensely investigated for its therapeutic potential in disorders of appetite and abnormalities of body weight. Hormones involved in glucose and adipose homeostasis, such as leptin and insulin, have been investigated as candidates for ghrelin regulation. While there have been many studies on the regulation of ghrelin secretion, the results have been variable and none have addressed the direct regulation of secretion from ghrelin producing cells at the molecular level. To understand the direct regulation ghrelin at the molecular level, we generated transgenic mice in which expression of enhanced green fluorescent protein (EGFP) under the control of the endogenous ghrelin promoter, allows the isolation of a pure population of ghrelin producing cells. These mice were healthy, fertile and displayed no phenotype compared to their WT littermates. Anti-ghrelin immunofluorescence studies in this transgenic mouse line confirmed that EGFP is faithfully expressed only in the ghrelin producing cells throughout GI tract. Dispersed single cells obtained by collagenase/EDTA digestion of gastrointestinal mucosa were subjected to fluorescence-assisted cell sorting (FACS) to produce a pure population of ghrelin producing cells. A variety of potential ghrelin secretagogues and inhibitors were applied to the acutely cultured pure ghrelin cells and their affect on ghrelin release measured by immunoassay for acylated ghrelin. These studies provide evidence for the direct stimulation by glucagon and inhibition by leptin of ghrelin secretion. Having a pure population of ghrelin-positive cells should facilitate our ability to interrogate the gene expression profile for potential candidates involved in the regulation of ghrelin secretion. In addition, the faithful expression of EGFP by ghrelin producing cells indicates that this promoter region can be further employed to generate both tissue-specific gain or loss-of-function transgenic mouse models for future studies.